Pathogenic bacteria cause numerous debilitating diseases in the US and around the world. Diverse modes of resistance shield bacterial pathogens from the function of even the most recently developed antibiotics, thereby prolonging infections, elevating treatment costs, and increasing the rate of patient mortality. Unfortunately, traditional antibiotics further drive resistance by targeting bacterial cell viability and growth. promising alternative is the development of antibiotic classes that target mechanisms of infectivity and virulence rather than cell viability. One prominent class of regulators recently associated with bacterial infections is the Hfq-binding small RNAs (sRNAs). Small molecule inhibitors against these sRNAs have the potential to provide a paradigm-changing approach to new antibacterial agents that will limit resistance, and also will have a significant impact as novl probes for the characterization of sRNA pathways. The overall objective of this application is to develop and validate assays for a high-throughput screen (HTS) of small molecule inhibitors of Hfq-binding sRNAs in bacteria. This will be accomplished through the following: Specific Aim 1: Develop primary assays to screen for small molecule inhibitors of the Hfq-binding sRNA Spot 42 in Escherichia coli. The primary assay will rely on Spot 42, although the end goal is to identify inhibitors of global sRNA activity. This aim will be accomplished through the construction of a GFP reporter system targeted by heterologously expressed Spot 42, and determination of the statistical parameter Z' to assess the robustness of the assay. Specific Aim 2: Configure the assay for high-throughput screening of small molecule inhibitors. The assay will be adapted to a 1536-well high-throughput format. A pilot screen of 10,000 compounds then will be conducted to validate the HTS-readiness of the primary assay, followed by dose-response analysis of re-synthesized putative hit compounds from the pilot screen. Specific Aim 3: Establish secondary assays to validate the activity of library hits and their potential antibacterial function. The collection of secondary assays based on reporter and phenotypic measurements will identify non-cytotoxic compounds that specifically inhibit global sRNA signaling in bacterial pathogens. The long-term goal is to address pressing challenges in medicine by better understanding and exploiting RNA regulators. The development of high-throughput assays to identify small-molecule inhibitors of Hfq-binding small RNAs in E. coli is directed toward this goal. The research is highly innovative because it is based on a completely novel approach to study RNA regulators in bacteria and to treat bacterial infections. The work represents an interdisciplinary effort between the labs of the PI and co-I, which leverages the PI's expertise in characterizing Hfq-binding small RNAs in bacteria and the co-I's discovery of the first small molecule inhibitors of microRNAs through the development of an HTS in mammalian cells.